Fig. 6. Role of IGF-IR in the sensitization of MCF-7 and T-47D cells to PD098059. A, MCF-7 and T-47D cells were plated in triplicate in 24-well plates at densities of 1 x 104 and 2.5 x 104 cells/cm2, respectively. Cells subsequently were incubated in complete growth medium supplemented with control antibody/DMSO, control antibody/PD098059, anti-IGF-IR antibody/DMSO, or anti-IGF-IR antibody/PD098059. After 4 days, triplicate wells were counted for each treatment (Table 1) . The number of cells in antibody/PD098059-treated wells was expressed as a percentage of the number of cells in the respective antibody/DMSO-treated wells. Means are shown; bars, SD. The Student’s paired t test was used to determine that PD098059-inhibited growth in the presence of anti-IGF-IR blocking antibody was significantly different from PD098059-inhibited growth with control IgG antibody. *, P < 0.05. Data shown are representative of three similar experiments. B, MCF-7 and T-47D cells were plated at equivalent subconfluent densities and were treated as in A or were left untreated. After 3 h, cell lysates were prepared with RIPA buffer, and 750 µg of lysate protein were immunoprecipitated (IP) with polyclonal anti-IGF-IR, resolved by SDS-PAGE, and immunoblotted with anti-phosphotyrosine (anti-pTyr). From the same lysates, 20 µg of protein were fractionated by SDS-PAGE and immunoblotted with anti-phosphoERK1/ERK2. The membranes were stripped and reprobed with anti-IGF-IR or anti-ERK2 to confirm that equivalent amounts of protein were present in each lane.